Dihydrofolate reductase from chicken liver binds the dye Cibacron Blue F3GA in an "ionic mode" and the dye is displaced only by methotrexate, folate or dihydrofolate but not by NADP(H). The enzyme from Lactobacillus casei, however, binds the dye in a nonpolar mode; only a combination of methotrexate (or folate or dihydrofolate) and NADP(H) can displace the dye completely from the enzyme. The dihydrofolate reductase catalytic reaction using NADPH as the coenzyme is activated by NaC1 but the reaction using NADH as the coenzyme is inhibited by NaC1. This dichotomy in the kinetic behavior is due to the mutual exclusivity in the binding of NADPH and chloride and mutual accomodation of NADH and chloride, as revealed by 35C1 NMR studies.